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The mass-production capability of hybridoma technology is bottlenecked by the routine screening procedure which is time-consuming and laborious as the requirement of clonal expansion. Here, we describe a 1-day chemiluminescent screening protocol for specific hybridoma cells on conventional 96-well plate via a proximity-rolling circle activated enzymatic switch (P-RCAES) strategy. The P-RCAES uses a pair of antigen-DNA probes to recognize secreted specific antibody and proximity-induce rolling circle amplification for mass-production of pyrophosphate to activate Cu(II) inhibited horseradish peroxidase and generate a strong chemiluminescent signal. The P-RCAES based homogeneous chemiluminescent assay can detect antibody down to 18 fM, and enables the screening of specific hybridoma cells secreting PCSK9 antibody at single-cell level without tedious cloning process. The proposed fast screening protocol has good expansibility without need of sophisticated instruments, and provides a screening method for greatly improving the efficiency of hybridoma technology.

Chemiluminescent screening of specific hybridoma cells via a proximity-rolling circle activated enzymatic switch